Method and apparatus for sorting cells

ABSTRACT

Apparatus for sorting and orienting sperm cells has a pair or walls in confronting relationship forming a flow chamber having inlet, a downstream outlet, and intermediate detector region. The inlet receives first and second spaced apart streams of input fluid and a third stream of sample fluid containing the cells to be sorted. The first and second streams have respective flow rates relative to third stream, such that the third stream is constricted forming a relatively narrow sample stream, so that the cells are oriented parallel to the walls. A detector detects desired cells and a sorter downstream of the detector for sorting the desired cells from the stream.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 11/046,896 filed Feb. 1, 2005 now U.S. Pat. No.7,355,696.This application is a reissue application of U.S. Pat. No.7,545,491, issued Jun. 9, 2009, and is a divisional U.S. applicationSer. No. 11/046,896, filed Feb. 1, 2005, now U.S. Pat. No. 7,355,696,issued Apr. 8, 2008 which is incorporated herein by reference in itsentirety.

The invention pertains to a flow sorter employing a multiangulardiscriminating detection and imaging system, for sorting cells. Anotheraspect of the invention pertains to a method and apparatus for opticaldetection and for imaging of objects.

Known imaging systems tend to be azimuthally symmetric, accepting lightwithin a certain range of angles established by the numerical aperture,(NA) of the imaging system. All light coming from the object planewithin the NA is ideally transferred to the imaging plane uniformly, inthe absence of aberrations or vignetting by optics or apertures whichare too small. The reason for this design is that it is desirable tohave a reasonably high light collection efficiency (i.e. a high NA) andthe angular variations in intensity often do not carry importantinformation.

An exemplary imaging system is a single round lens or a pair of roundlenses. For cases where high collection efficiency is desired, such asin imaging systems which are dim, a high NA system is designed by usingan optical element which is large compared to the size of the object,and which is close compared to its size. In this way, the lens capturesa large fraction of the light. High NA is also important for maximizingresolution and for obtaining a narrow focal depth.

Flow cytometers are devices which use optical scattering andfluorescence to discriminate between cells or other small objects suchas fluorescent beads, and to sort them based upon the discriminatedoptical measurements. As objects stream through a narrow jet, inputlaser light scatters, impinging on the objects, and incitesfluorescence. Scattered and fluorescent light signals are detected atvarying angles to characterize and discriminate objects with differingproperties.

One difficulty in gender sorting sperm is the very flat shape of sperm,especially bovine sperm. The flat shape, combined with the higher indexof refraction of DNA relative to the aqueous environment, causes lensingof light and internal reflection, including fluorescent light whichoriginates in the sperm head. This lensing causes light to be emittedpreferentially through the edges of the sperm, with much lower emissionthrough the two flat faces of the sperm head. Thus, detection of lightintensity and determination of X or Y chromosomal content of the spermis dependent upon reliable alignment of the sperm and the ability toview the sperm fluorescence from multiple angles.

Known alignment systems employ a device in which speira cells areoriented and sprayed into a detection zone by means of a nozzle such asillustrated in Rens et al., U.S. Pat. No. 5,985,216. In such a device,the sorting nozzle has an elliptical cross section for orientingflattened cells. A disadvantage of Rens is that if the flow rate isabove about 5000 sperm cells per second, the cells can not be reliablyimaged and characterized. A 5000 sperm cells per second sperm flow rateis inefficient and time consuming. A more practical rate for spermsorting is around 100,000 sperm cells per second or higher.

One type of imaging system used to manipulate small particles isdescribed in U.S. patent application Ser. No. 10/974,976, entitled“SYSTEM AND METHOD FOR MANIPULATING AND PROCESSING NANOMATERIALS USINGHOLOGRAPHIC OPTICAL TRAPPING”, filed Oct. 28, 2004, and in U.S. patentapplication Ser. No. 10/934,597, filed Sep. 3, 2004, entitle “MULTIPLELAMINAR FLOW-BASED PARTICLE AND CELLULAR SEPARATION WITH LASERSTEERING”, the teachings of both, which are herein incorporated byreference.

SUMMARY OF THE INVENTION

The present invention pertains to a flow sorter employing a multiangulardiscriminating detection and imaging system, for sorting cells. Oneaspect of the invention pertains to a method and apparatus for opticaldetection and for imaging of objects. In particular, the presentinvention is directed to a method and apparatus for characterizing andsorting bovine sperm by gender. However, it should be understood thatother types of mammalian sperm cells and the like may be sorted by usingthe present invention.

The present invention is based upon the discovery that a flow sorter forsorting and orienting cells employs a flow channel having an inlet, anoutlet, an intermediate detection zone, and optionally, a sortingregion. The inlet receives one each of alternating spaced streams ofinput sheath fluid and a sample stream containing the cells to be sortedbetween sheath streams. The sheath streams and the sample stream haverespective flow rate or pressures in the flow chamber such that thesample stream is constricted thereby forming a relatively narrow samplestream in the detection region whereby the cells are oriented in aselected direction relative to the input light. A detector employing amulti angle or K-Vector imaging setup is focused in the detection zonefor discriminating between desired and undesired cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow diagram of steps for sorting cells which have been dyedwith a fluorescent dye.

FIG. 2 is a perspective schematic illustration of a flow device orcartridge according to the invention.

FIG. 3 is a schematic illustration of a single channel flow device forsorting.

FIG. 4 is a side-view of a multichannel flow device for sorting.

FIG. 4A is a top plan (edge) view, facing the inputs, of themultichannel system shown in FIG. 4.

FIG. 5 is a schematic representation of a single channel sensor forsensing scattered light (i.e., optical system for K-vector imaging).

FIGS. 5A-5D illustrate alternative optical elements employed in thearrangement of FIG. 5.

FIG. 6 is a schematic representation of a single channel sensor fordetecting fluorescent and scattered light using K-vector imaging.

FIG. 7 is a multichannel channel device for K-vector imaging withexcitation and detection of scattered and fluorescent light.

FIG. 8 is a schematic representation of an external actuator adjustingflow speeds in a channel or device for sorting cells.

FIG. 9 is a schematic representation of a top view of a multichanneldevice or cartridge for detecting and sorting cells.

FIGS. 10A-10D show various alternative ways of steering cells usingthree actuators on M, W, F (FIG. 10A), two actuators on S1, S2 (FIG.10B), one actuator on S1 (FIG. 10C), and two actuators on M, F (FIG.10D).

FIGS. 11A-11B show various alternative ways of killing cells by laserkilling or activation (FIG. 11A), and electrical killing or activation(FIG. 11B).

DESCRIPTION OF THE INVENTION

FIG. 1. illustrates a flow diagram setting forth the steps forcharacterizing, sorting and processing objects, for cryogenicpreservation, particularly bovine sperm cells.

The first stage from collection 100, extension 101, to slow cooling 102,is the subject of various procedures, some of which are novel and othersof which are known.

A novel system for preparing cells for sorting is set forth in copendingU.S. patent application Ser. No. 11/048,101, entitled: “Novel Method ForIn Vivo Staining of Cells for Identification and Soiling”, filed on Feb.1, 2005, the teachings of which are incorporated herein by reference.

The steps include loading a sample into a disposable chip 200; filteringthe sample to remove large aggregate material 201, such as yolkaggregates; employing flow based alignment 202 as set forth hereinafter;employing parallelized gender detection 203, discrimination (i.e.,gender discrimination 204) and actuation (i.e., gender actuation 205)steps; passive concentration and balancing 206, and delivery to anoutput reservoir 207. The method may also optionally eliminate somesteps and include a discrimination and killing step for removingunwanted live sperm.

The gender sorting steps 103 which includes the above, are then followedby slow cooling to 4° C. and settling 104; final extension 105; packingin straws 106; settling 107 and freezing 108 steps.

FIGS. 2-3 illustrate in various folins a flow cytometer 300 according tothe invention. The device may be a single channel system. However, theinvention is well suited for multichannel applications, particularly insperm cell sorting applications, where large numbers of sperm cells mustbe sorted in a reasonable amount of time.

In FIGS. 2-3, the device 300 comprises a body or flow chamber 312 formedof a pair of confronting walls 314 and end walls 316 (only one of whichis shown in FIG. 1), an open top or input 318 and open bottom or output320.

The input 318 is divided into three sections including outboard inputs322 and central or sample input 324. The outboard inputs 322 are forreceiving a sheath fluid 326 therein and the central input 324 is forreceiving a sample fluid 328 containing a liquid medium and cells 330dispersed therein.

The output 320 has outboard output sections 332 and central samplecollection channels, namely left output sample channel 334L, centraloutput sample channel 334C and right output sample channel 334R. Channel334L is for a first sorted sample, 334C is for a second sorted sample,and 334L is for yet another sorted sample.

Sheath fluid 326 is input at outboard inputs 322 at a selected flowrate. Sample fluid 326 is introduced in central input 324 at a selectedflow rate or pressure relative to the sheath flow rate or pressure suchthat the sheath fluids compress and constrict the sample flow to arelatively narrow sample flow path 336 as shown. In an exemplaryembodiment, the width of the sample flow path 336 is about 10% or lessof the width of the sample fluid at the central input 324, e.g. about 50microns.

The cells 330 are circular but flattened. As a result, constriction ofthe sample fluid causes the cells 330 to orient themselves so that theirflat sides are roughly parallel to the confronting walls 314. Theintensity of light radiated by a cell is different at differentorientations. So to compare the intensity of two or more cells, theymust have the same orientation. Thus, aligned cells reduce noise orsystematic error caused by having anisotropic light emitter at randomorientations.

Alternating inputs of sheath fluid 326 and input sample or objectsolution 328 (see FIG. 3) enter the system, creating a small amount ofconstriction (relative to the constriction in the orthogonal direction)401, which causes shear and the shear flow aligns the cells 400. Thisalternating flow pattern is squeezed more severely between two longinput sheath fluid flows, which achieves the necessary alignment. Thisarrangement may be combined with detection hereinafter to provide aparallel system where multiple flows may be interrogated.

Specifically, the constricting flow moves objects into the focal plane402, and accelerates movement through the detection region 403. Thecurve 404 in the system shows the fluid boundary 404, and the detectionregion 405 allows for characterization. The light cone 406 allows forinterrogation, and the default stream position 407 can be steeredbetween multiple outlets.

By varying the flow rate through the three output channels 409-411,cells or other objects in the solution can be sorted into one ofmultiple output streams. The actuation may be done in various ways, asenumerated above. High-speed flow switching may be performed by piezodevices which may be intrinsic to the machine, or intrinsic to thedisposable flow channel cartridge. Flow switching region 408 controlsthe precise flow rate, which varies over time to switch between outputchannels 409-411 (where V2<V1, and V4˜v2).

The detector 340 (FIG. 2) comprises a laser 342 or other suitable sourceproducing an output beam 344 directed towards the sample flow path 336in a detection zone 338 intermediate the input and output. The beam 344impinges on the cell 330 in the detection zone 338 and scatters formingan output beam 346. The cell contains a fluophor and thus producesfluorescent light as well which is contained in the output beam 346.Respective scattered and fluorescent components 346S, 346F of the outputbeam 346 are input to an optical detector containing an optical system348 and an electronic detector system 350. The optical system andelectronic detector system are discussed hereinafter.

The output beam 346 carries information to the detector 340 whichdiscriminates among the cells 330 and produces an output 354 to a sorter356. The sorter 356 controls drivers 358 in operative relationship withthe output channels 334 in order to vary the relative flow rates suchthat the each cell 330 is sorted into a proper channel. Alternatively,the cells may be sorted as wanted or unwanted, and the wanted cells maybe collected and the unwanted cells may be destroyed.

FIG. 3 shows a single channel system having only two sheath flows 326and one sample flow 328. FIG. 4 shows a multi-channel system with foursample flows 328 and shared sheath flows 326. FIG. 4A shows the flowchamber and flow paths in top plan.

FIGS. 4-4A show one possible way of parallelizing the design to havemany parallel streams 500 of input solution. Flow may constrict both inthe plane and normal of the plane. However, for eases where alignment ofcells is necessary, such as with bovine sperm, shear along the directionof the incoming light must be much larger to guarantee alignment normalto the incoming light. The optical investigation region 502 is wherelaser scattering and fluorescence takes place.

In the example, each sheath has a dedicated lens system, multiple PMTelements each of which is dedicated to a corresponding stream. It shouldbe understood that the system may have one lens detector system for allchannels and one laser optic system for all channels as well. There aremultiple possible output channels 503 for each flow stream.

A simplified plan view of the optical system of the detector 640 isshown in FIG. 5. A sample 660 lies in an object plane 662. In thisillustration output beam or, light rays 646 emanate from the sample 660as beamlets 646C, 646L, 646R. The beamlets 646C represent centrallyclustered beams near the central optical axis C; and beamlets 646L and646R are clustered to the left and right of the central axis C. Thebeamlets provide different views of the sample 660. A collection lens664 (which may be an objective lens from a microscope) is positioned adistance from the object plane 662 which is equal to the effective focallength (EFL) of the lens. The lens 664 may be a compound lens ifdesired, but for simplicity it is described it as a single lens with anEFL. By positioning lens 664 thus, light 646 from the object exits thecollection lens 664 is collimated as beamlets 666 likewise divided as666R, 666C, 666L as shown. This creates an infinity space in the imagingsystem. While the present invention does not require this infinityspace, it is a convenient arrangement. The lateral positioning of eachcollimated light ray is determined predominantly by its angle comingfrom the object plane. Beamlets 666L, 666R and 666C follow respectivebeamlets 6421, 642R and 642C.

Central light beamlets 666C exit lens 664 along central axis C tofocusing lens 676C which focuses the light on forward image plane 678C.Mirrors 672L, 672R separate off-axis beamlets 666L and 666R exiting thecollecting lens 664. Note that this may also be done with the placementof detector 674A (FIG. 5A) or optical fiber 674B (FIG. 5B) in thisregion which are small compared to the size of the beam in this space.Note also that additional optical elements may be inserted in thisspace, such as additional lenses 674C (FIG. 5C) or a pinhole 674D (FIG.5D) to constrain the range of angles of light coming out of thecollecting lens 664, or to control (restart or expand) the focal depthfrom which light is collected as in confocal microscopy measurements. Insome circumstances, a pair of additional lenses, with a pinhole, may beused. In other cases, a mask with controllable size, shape, or positionmay be used to control the light reaching a given detector.

The light from beamlets 666L is deflected by mirror 672L to leftfocusing lens 676L; and from beamlet 666R light is directed by mirror672R to lens 676R and right image plane 678R. Light detectors 680L, 680Rand 680C may be located in respective image focal planes 678L, 678R and678C to detect the respective images. These light detectors may be CCD,photo diodes, photomultiplier tubes, multi-anode photomultiplier tubes,or other sensors.

In many cases, it is desirable to collect both scattered light andfluorescent light, where at least one of the images or detections maderequire a reduced range of ray angles from the sample. FIG. 6illustrates how this may be done, using the K Vector Imaging setup asdescribed above, but with filters and additional beam splitters asnecessary. Similar elements have the same reference numerals.

In FIG. 6, illumination and excitation light 800 passes throughcollecting lens 801, and is reflected by mirrors 802, 803 throughemission filters 688EL, 688ER to focusing lenses 804, 805 and to photodetectors 690L, 690R, respectively, which form the left and rightfluorescent image planes.

The beamsplitter in 686C redirects light in the central field 666Cthrough an emission filter 688E to focusing lens 676FC. The output 689Eof emission filter 688E corresponds to fluorescence emission from thecell. Laser line filter 688L in the central optical axis filtersscattered laser light to lens 676C and photo detector 690C, where isformed the forward scattered image plane.

To improve the throughput and overall capabilities of a device,parallelization is desired. FIG. 7 shows how this is accomplished. Laser642 produces input laser beam 644, which excites fluorescence, is brokenup into N multiple beams by beamsplitter 690 (e.g., diffraction gratingor hologram). Beams 692A and 692N fan out and each are directed to thedetection zone of a corresponding sample sheath (see FIG. 4). Thebeamlets 692A-692N are collimated into parallel beams by the collimatinglens 694. The output 696 of the collimating lens 694, is directedthrough cylindrical lens 698 which focuses individual beams 700A-700Nonto the sample streams in the object plane 701. As shown, (FIG. 4) theinput stream is broken up into many streams. Light from one or allbeamlets 700A-700N in the example shown and sample streams enterscollecting lens 702K and is handled as described in FIG. 6 above. Here,however, detector array devices (such as multi-anode PMTs) device 720(720C, 720R, 720L, 720CF . . . ) one for each group of detection beamsare used. Detection is best accomplished not by placing a singledetector in each imaging position, but by using array detectors, such asa 32-element linear PMT array 720.

The throughput of a system which images or make measurements on manyobjects will depend, in part, upon the number of detectors and theirspeed. For many applications, a single detector such as photomultipliertube (PMT) is used in each image plane. This is suitable for cases whereobjects pass through the object plane in a single-file line.

The sorter 856 is hereinafter described in detail. A single channelsorter is shown in FIG. 8. The sorter 856 comprises a structural member812; a channel defining layer 816 formed with a slot 818 defining a flowchannel 820; a flexible membrane layer 822 atop the channel defininglayer 816, and a structural member 824 completing the arrangement.Piezoelectric or another type actuator 826 is coupled to a controller828. A piston 860 driven by actuator 826 engaged flexible membrane 822opposite the flow channel 820 to change the flow pattern within thechannel in accordance with the voltage supply to the piezo-electric. Aplurality of such structures may be miniaturized so as to be located inthe actuator window whereby multiple streams of samples may be sorted.

The one or more actuators 826 may include: a piezo-electric transducerfor converting electrical signals into mechanical actuation of the flowrates; a thermal heater for heating a region to quickly expand a fluid,material, or bubble; a thermal bubble generation for creation of abubble to reduce the flow of the solution; a capacitive motion devicefor a membrane; an optical device for heating or moving material, wall,membrane, bubble, or other material or object to impact the flowvelocity into one or more of the output channels 934. The actuation maybe intrinsic to the device or may be externally applied. For example,the actuator 826 and piston 860 may be external equipment, separate fromthe disposable flow device 856 (i.e., disposable chip withnon-disposable/external actuator).

FIG. 9 illustrates the parallel arrangement of multiple channels inwhich input channels 900 feed the sheath streams to the flow channelsand output channels 901 receive the various sorted sperm cells. Window930 is provided for coupling interrogation light to each of the paralleldetector sample channels. Sorter window 932 receives a plurality ofactuators and disposable sorter elements. Chip registration pins of thedevice 100 are designated as 940.

FIGS. 10A-10R illustrate alternative embodiments for steering cells,employing a variety of actuators 934 in a flow device for sorting sperm.In each case, one or more actuators are used, where each actuator couldbe based off of piezoelectric devices (intrinsic or extrinsic to thecartridge), capacitive actuators, thermal expansion, or othertechnologies as described in the prior disclosures. In most cases, atleast two actuators 934 are desired, and possibly more than twoactuators, to control which exit channel 936 a particular cell or objectgoes into. The actuators allow one to control and vary the flows at veryhigh rates. Only minor perturbations to the flow are necessary to causea stream to temporarily move from one exit to another.

FIGS. 11A-11B show examples of killing or activation setups withoutsorting for achieving a desirable result. In the FIG. 11A, a laser 940impinges the object/sperm stream 942. This laser is controlled to onlyimpinge lethal energy on certain sperm or objects depending upon theresult of the interrogation. For example, this laser may kill certainsperm or other cells as desired. For example, it may kill all sperm of agiven or uncertain gender. Alternately, the laser may not directly killthe cells but may otherwise “activate” them. For example, it couldactivate some chemical which has been previously introduced into thesperm, having the overall result of killing them or otherwise impairingfertilization. In more general applications, activation may have a muchbroader range of activities.

FIG. 11B shows killing or activation using lethal electrical pulses,introduced through electrodes 944 which are inserted into the flow inorder to locally access the central stream 942. As with the lasersolution, the electrodes can be quickly pulsed on or off depending uponthe result of the interrogation.

Steering may also be achieved optically where the cells are manipulatedby an optical trapping apparatus. Alternatively, the actuation processmay be electroporation of the cells, which may be lethal or have othereffect on the cells.

The following technique aligns sperm cells using squeezing flow:

Three flows were fed into a flow chip using a peristaltic pump. Eachflows were kept in laminar regime so that each flow does not mix eachother. The stream containing sperms flows between top and bottom streamswhich are waters. While the velocity of top and bottom flow is keptsame, by changing the ratio of those to the sperm flow, we could see thesqueezing of sperm flow.

sperm orientation (%) angle from flow direction (degree) Re <3 <15<45 >45 .066 51.35 29.73 13.51 5.41 1.35 61.11 14.81 22.22 1.85 2.1366.67 16.67 16.67 0

It is expected that the squeezed flow helps the sperm oriented to theflow direction.

Images of the sperm in flow were taken using a CCD camera equipped onour microscope.

Above table shows the degree of sperm orientation in the flow where Reis the Reynolds number defined as Dur/m where D is diameter of flowchannel, U is the speed, r is density of fluid and m is the viscosity.

Re indicates whether the flow is laminar or not, even though Re below1000 is considered laminar flow, in some applications, very small Resuch as below 1 is required.

As the speed of sperm flow increases the Re in the channel inletincreases but still remains in laminar region indicating the flow streamis not disturbed in our experimental region.

The orientation of sperm was quantified by numbering of those asfunction of degree alignment of sperm head to flow direction.

In the experiment range, about 80% of sperms imaged were oriented inless than 15 degree to flow direction.

Better alignment to flow direction was shown at higher speed but moredisturbed sperms were also found.

The results shows that the system could align sperms using thistechnique.

It should be understood that temperature control of the sheath fluid andsample fluid can be employed to prolong sperm life. In an exemplary,embodiment the temperature of the fluids in the flow device may bemaintained around 2-10° C. in order immobilize the sperm cells andthereby extend their lifetime.

We claim:
 1. A sensor for imaging an object in an object planecomprising: an optical source for illuminating the object using k-vectorimaging; a collection lens provided a predetermined distance from theobject plane equal to an effective focal length of the collection lens;wherein the illumination of the object produces beams which exit thecollection lens and are collimated as beamlets which are divided to formmultiple images of the object in the form of light leaving the objectplane at different angles relative to a central axis of the sensor; andwherein a lateral positioning of each collimated beamlet is determinedprimarily by its angle from the object plane; and a detector responsiveto light from the object.
 2. The apparatus of claim 1 wherein thedetector is responsive to at least one of light directed along theoptical axis of the sensor and light directed off the axis of thedetector.
 3. The apparatus of claim 2 wherein the off-axis detectorcomprises at least two photo detectors laterally displaced with respectto the central axis for detecting off-axis light corresponding todifferent positions in the object plane.
 4. The apparatus of claim 3further including a filter for filtering at least one of fluorescent andscattered light from each beam.
 5. The apparatus of claim 3 wherein thephoto detector comprises a multiple element photo multiplier tube fordetecting multiple beams of light.
 6. The apparatus of claim 1 whereinthe optical source comprises a laser.
 7. The apparatus of claim 6further including a lens for collimating the light from each object intoseparate beams.
 8. The apparatus of claim 7 further including collectinglens for each beam.
 9. The apparatus of claim 7 further including asplitter for splitting on-axis light from off-axis light.
 10. Theapparatus of claim 1 further including a cell killer downstream of thedetector for killing undesired cells.
 11. The apparatus of claim 10where the cell killer comprises at least one of a laser and electrodefor directing lethal energy at the undesired cells.
 12. The apparatus ofclaim 10 wherein the cell killer comprises a source of light foractivating a lethal target in the cell.
 13. The apparatus of claim 1further including a cell electroporation for effectively processing orkilling undesired cells.
 14. The apparatus of claim 1 wherein the flowdevice is operable at about 2° C.-10° C. to prolong cell life.
 15. Asensor for imaging an object in an object plane comprising: an opticalsensor using k-vector imaging; a beamsplitter that breaks an input beamfrom the optical source into multiple beams; a first collimating lensthat collimates the multiple beams from the beamsplitter into parallelbeams; wherein the parallel beams are focused onto the object in asample stream in the object plane or onto multiple objects in multiplestreams in the object plane; a collection lens provided a predetermineddistance from the object plane equal to an effective focal length of thecollection lens; wherein an illumination of the object produces beamswhich exit the collection lens and are collimated as beamlets which aredivided to form multiple images of the object in the form of lightleaving the object plane at different angles relative to a central axisof the sensor; and wherein a lateral positioning of each collimatedbeamlet is determined primarily by its angle from the object plane and adetector responsive to light from the object.
 16. The apparatus of claim15, wherein the detector is responsive to at least one of light directedalong the optical axis of the sensor and light directed off the axis ofthe detector.
 17. The apparatus of claim 16, wherein the off-axisdetector comprises at least two photo detectors laterally displaced withrespect to the central axis for detecting off-axis light correspondingto different positions in the object plane.
 18. The apparatus of claim17 further including a filter for filtering at least one of fluorescentand scattered light from each beam.
 19. The apparatus of claim 17wherein the photo detector comprises a multiple element photo multipliertube for detecting multiple beams of light.
 20. The apparatus of claim15 wherein the optical source comprises a laser.
 21. The apparatus ofclaim 20 further including a lens for collimating the light from eachobject into separate beams.
 22. The apparatus of claim 21 furtherincluding collecting lens for each beam.
 23. The apparatus of claim 21further including a splitter for splitting on-axis light from off-axislight.
 24. The apparatus of claim 15 further including a cell killerdownstream of the detector for killing undesired cells.
 25. Theapparatus of claim 24 where the cell killer comprises at least a laseror at least an electrode for directing lethal energy at the undesiredcells.
 26. The apparatus of claim 24 wherein the cell killer comprises asource of light for activating a lethal target in the cell.
 27. Theapparatus of claim 15 further including a cell electroporation foreffectively processing or killing undesired cells.
 28. The apparatus ofclaim 15 wherein the flow device is operable at about 2° C.-10° C. toprolong cell life.
 29. A sensor for imaging an object in an object planecomprising: an optical sensor using k-vector imaging; a collection lensprovided a predetermined distance from the object plane equal to aneffective focal length of the collection lens; wherein the object is ina sample stream between at least two sheath streams and wherein thesample stream and the at least two sheath streams have respective flowrate or pressures in a flow chamber such that the sample stream isconstricted in a detection region of the flow chamber whereby the objectis oriented in a selected direction relative to a beam from the opticalsource; wherein an illumination of the object produces beams which exitthe collection lens and are collimated as beamlets which are divided toform multiple images of the object in the form of light leaving theobject plane at different angles relative to a central axis of thesensor; and wherein a lateral positioning of each collimated beamlet isdetermined primarily by its angle from the object plane and a detectorresponsive to light from the object.
 30. The apparatus of claim 29,wherein the sheath streams flow at a relatively faster flow ratecompared to the flow rate of the sample stream, whereby a pressure isexerted from the sheath streams towards the sample stream.
 31. Theapparatus of claim 29, wherein the sheath streams flow at a relativelyslower flow rate compared to the flow rate of the sample stream, wherebya pressure is exerted from the sheath streams towards the sample stream.32. A sensor for imaging a cell in an object plane comprising: anoptical sensor using k-vector imaging; a collection lens provided apredetermined distance from the object plane equal to an effective focallength of the collection lens; wherein an illumination of the cellproduces beams which exit the collection lens and are collimated asbeamlets which are divided to form multiple images of the object in theform of light leaving the object plane at different angles relative to acentral axis of the sensor; and wherein a lateral positioning of eachcollimated beamlet is determined primarily by its angle from the objectplane; a detector responsive to light from the object; and a cell killerdownstream of the detector for killing or damaging undesired cells. 33.The apparatus of claim 32 wherein the cell killer comprises at least alaser or at least a cell electroporation for directing lethal ordamaging energy at the undesired cells for effectively processing orkilling undesired cells.